| 题名 | 活细胞单分子实时显示的光学途径探索 |
| 作者 | 王琛 |
| 学位类别 | 博士 |
| 答辩日期 | 2005 |
| 授予单位 | 中国科学院上海光学精密机械研究所 |
| 导师 | 徐至展 |
| 关键词 | 单分子 取向 全内反射荧光探测系统 精确定位 活细胞 受体蛋白 双光子吸收截面 |
| 其他题名 | The investigation of optical methods for the real-time imaging of single-molecule in living cell |
| 中文摘要 | 自20世纪90年代以来，随着微电子学、光电技术、纳米科技的飞速发展，科学的进步到了一个新的起点，不但要求对整体特征的正确理解，同时要求对个体间差异的详细阐述。人们力图在单分子水平上捕捉细胞生命活动的细节；力图实时检测化学反应过程中单分子的形态和行为，克服集团平均研究的缺陷，在更深入的层次揭示化学反应的规律；力图了解单个分子本身的物理特性以及与周围环境的相互作用，揭示物理本源。在这样的大环境下单分子探测科学应运而生。本论文工作按照国家973课题"活细胞单分子实时视见研究"的要求，重点是寻求"可实时视见地观察细胞生命活动过程中的单分子之间的相互作用"的光学探测途径。论文对当今国际上单分子研究的现状、探测方法、在生物学上的新兴应用和发展前景进行了较全面的调研及评述。围绕单分子全内反射荧光探测技术，开展了三个方面的研究工作。一，全内反射成像理论的研究。二，全内反射荧光探测系统的建立及应用研究。三，超短脉冲下的双光子吸收截面探测系统的建立以及应用研究。概括起来，取得了以下几个方面的创新性进展：1．隐失场偏振理论的发展。（a）对不同偏振光入射下的隐失场偏振特性进行了严格的推导，并得到了隐失场激发下的荧光发射各向异性的定量分布；（b）基于隐失场的偏振特产胜，提出了结合落射式荧光和全内反射荧光两种成像方式，通过对荧光强度的数值分析来确定单分子三维取向的新思路。我们的方法具有光路易于调节、数据处理简单的特点。2。个内反射超分辨理论的发展。（a）我们分析了将驻波激发引入到全内反射成像力一法中后，各种入射条件对成像分辨率的影响，给出了提高分辨率的址优化原则；（b）提出利川隐失场独特的纵向成像特性，可以在单分子的荧光探测中直接快速的确定单分子荧光团间的纵向间隔和每个荧光团的纵向绝对位置，即实现单分子的纵向超分辨荧光成像。3．建立了一套物镜型全内反射荧光成像系统，并解决了系统重要参数的标定问题，自行设计方案对系统分辨率、系统探测效率和光强计算等进行了细致的定量测量和分析。4．以此系统为平台进行了离体荧光索分子的探测，实现了平均精度为23nm的单分子空间定位。并首次在没有配体EGF刺激的情况下，研究了CHO活细胞内单个EGFR蛋白分子的膜动力学行为。不仅观察到量子态淬灭的单分J一典型特性，还观察到"两步阶跃"的动态行为，这将为研究EGFR的1聚化机制提供新的数据。5．建立了·套790n而13fs超短脉冲激发下的双光子吸收截面的测量装置，并首次对一种新型的对称型菊类有机染料进行了测量，结果显示其具有与非对称型菊类衍生物相同量级的大的吸收截面。以往双光子有机合成理论认为菊类染料的大的双光子吸收截面源于非对称型取代基之间的电荷转移，我们的测量则显示可能存在其它吸收机制，为研究双光子有机材料提供了重要参数。该系统同时为分子相互作用的荧光共振能量转移（FRET）光谱研究奠定了实验基础。 |
| 英文摘要 | In the recent decade, significant advances of microelectronic, photoelectric technique and nanotechnology have brought science into a new era during which not only exact understandings of ensemble representation are required, but also the detailed specific individuals are expected to be expatiated. Human beings are trying to capture rare events of celluar vital activity in single-molecule level; trying to obtain real-time detection of individual molecules and their time evolution in chemical reactions that had been buried in the average properties of a system under study; trying to explore physical properties of individual molecules together with their interacting surrounding nanoenvironment towards the physical origin. According to requirement of the grant from the Major State Basic Research Development Program of China entitled "The study of single-molecule visualization in living cell", the work in this thesis is to investigate method suitable for real-time single molecule detection in living cell. We give a general review on current researches and detection methods of single-molecule study, as well as emerging biological applications and future prospects. My work included three parts. One is the theoretical study of total internal reflection fluorescent imaging. Another is the experimental setup of TIR fluorescence microscope system and its application. And the last is the development of a system for the two-photon absorption (TPA) cross-section measurement and its application. There are several innovations in this work. 1. The polarized imaging theory of TIR fluorescence was developed, (a) The different evanescent fields made by excitations with different polarized incident fields have been rigorously derived, and their influence on fluorescence anisotropic emission has been discussed, (b) We proposed a new imaging method to determine the three-dimensional dipole moment orientation of single fluorophore by comparing fluorescence intensities measured with far-field epi-fluorescence imaging scheme and TIR fluorescence imaging scheme. The super-resolution imaging theory of TIR was developed, (a) The optimal condition for enhanced resolution in evanescent standing wave microscopy has been given through considering the influence of various incident conditions on the resolution, (b) Utilize unique intensity pattern in the z direction of evanescent wave, we point out that it is possible to calculate relative separation distances of individual molecules, and their axial position in single-molecule experiments. We setup a TIR fluorescence microscope system and the performance properties of it, including quantitative estimation of light intensity and light collection efficiency have been tested. Based on the microscope, optical detection of single fluorescein molecule has been achieved on the surface of polymethyl methacrylate (PMMA) film, as well as single EGFR molecules in living CHO cell have been visualized. In addition to the typical one-step and two-step quenching of single molecules, we also observe two-step rising dynamic phenomena which may provide new sight for the dimerization mechanism of epidermal growth factor receptor (EGFR). A set of TPA cross-section measurement system has been built using 13fs/790nm Ti: sapphire laser, and the measurement of a new fluorine-based dye is stated in detail.Over the past years, many asymmetric fluorine derivatives have been studied since it is considered that charge transfer between asymmetric substituents can result in strong TPA. Our data indicate that the charge transfer may not be the key process for TPA. |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.siom.ac.cn/handle/181231/15570]  |
| 专题 | 上海光学精密机械研究所_学位论文 |
| 推荐引用方式 GB/T 7714 | 王琛. 活细胞单分子实时显示的光学途径探索[D]. 中国科学院上海光学精密机械研究所. 2005. |
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